U.S. patent application number 10/525157 was filed with the patent office on 2006-07-20 for refined royal jelly.
Invention is credited to Shigeharu Fukuda, Toshio Miyake, Takashi Shibuya.
Application Number | 20060159834 10/525157 |
Document ID | / |
Family ID | 31973151 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060159834 |
Kind Code |
A1 |
Shibuya; Takashi ; et
al. |
July 20, 2006 |
Refined royal jelly
Abstract
The object of the present invention is to provide an
allergen-reduced royal jelly, having less fear of causing allergic
symptoms, obtained by reducing allergens and keeping the useful
pharmacological actions of royal jelly, a process for producing the
same, and its uses. The present invention solves the above object
by providing an allergen-reduced royal jelly where the amount of
water-soluble proteins is reduced to less than 50% (w/w) to the
amount of total proteins; a process for producing the
allergen-reduced royal jelly comprising the steps of separating a
royal jelly into a precipitate and a supernatant comprising
water-soluble proteins by adding water and centrifuging, collecting
low molecular substances by eliminating high molecular substances
comprising water-soluble proteins from the resulting supernatant
using ultra-filtration or gel filtration chromatography, and mixing
the resulting low molecular substances with the aforesaid
precipitate; a composition comprising the allergen-reduced royal
jelly; and the uses of the composition for foods, feeds, baits, pet
foods, and cosmetics.
Inventors: |
Shibuya; Takashi; (Okayama,
JP) ; Fukuda; Shigeharu; (Okayama, JP) ;
Miyake; Toshio; (Okayama, JP) |
Correspondence
Address: |
Browdy and Neimark
624 Ninth Street N W
Washington
DC
20001-5303
US
|
Family ID: |
31973151 |
Appl. No.: |
10/525157 |
Filed: |
September 4, 2003 |
PCT Filed: |
September 4, 2003 |
PCT NO: |
PCT/JP03/11311 |
371 Date: |
March 4, 2005 |
Current U.S.
Class: |
426/635 |
Current CPC
Class: |
A61Q 19/00 20130101;
A23G 9/38 20130101; A23K 20/10 20160501; A61K 8/988 20130101; A23L
33/15 20160801; A23L 21/20 20160801; A23V 2002/00 20130101; A23V
2002/00 20130101; A61K 8/64 20130101; A23K 50/40 20160501; A23V
2200/304 20130101; A23L 33/11 20160801 |
Class at
Publication: |
426/635 |
International
Class: |
A23K 1/00 20060101
A23K001/00 |
Claims
1. An allergen-reduced royal jelly, where the amount of
water-soluble proteins is reduced to less than 50% (w/w) to the
amount of total proteins.
2. The allergen-reduced royal jelly of claim 1, where allergens are
eliminated without substantially losing the useful pharmacological
action of royal jelly.
3. The allergen-reduced royal jelly of claim 1, where the weight
ratio of the total proteins to 10-hydroxy-2-decenoic acid, on a
free fatty acid basis, is less than six.
4. A process for producing an allergen-reduced royal jelly
comprising the steps of separating a royal jelly into a precipitate
and a supernatant comprising water-soluble proteins by adding water
and centrifuging; collecting low molecular substances by
eliminating high molecular substances comprising water-soluble
proteins from the resulting supernatant using ultra-filtration or
gel filtration chromatography; and mixing the resulting low
molecular substances with the aforesaid precipitate.
5. The allergen-reduced royal jelly of claim 1, which is obtainable
by the process of claim 4.
6. A composition comprising the allergen-reduced royal jelly of
claim 1 together with another component.
7. A composition produced by incorporating the allergen-reduced
royal jelly of claim 1 into an anhydrous saccharide.
8. The composition of claim 7, wherein said anhydrous saccharide is
any one of anhydrous trehalose, anhydrous maltose, and anhydrous
cyclic tetrasaccharide.
9. The composition of claim 6, further which comprises saccharide
transferred-vitamin C.
10. The composition of claim 6, further which comprises one or more
antioxidant substances.
11. The composition of claim 10, wherein said antioxidant
substances are flavonoids, polyphenols, vitamin E, and vitamin
C.
12. The composition of claim 6, which is in the form of a food,
feed, bait, pet food, or cosmetic.
13. The allergen-reduced royal jelly of claim 2, where the weight
ratio of the total proteins to 10-hydroxy-2-decenoic acid, on a
free fatty acid basis, is less than six.
14. The composition of claim 7, which comprises saccharide
transferred-vitamin C.
15. The composition of claim 7, which comprises one or more
antioxidant substances.
16. The composition of claim 15, wherein said antioxidant
substances are flavonoids, polyphenols, vitamin E, and vitamin
C.
17. The composition of claim 7, which is in the form of a food,
feed, bait, pet food, or cosmetic.
Description
TECHNICAL FIELD
[0001] The present invention relates to an allergen-reduced royal
jelly (Hereinafter, simply abbreviated as "ARRJ"), particularly,
ARRJ where the amount of water-soluble proteins is reduced to less
than 50% (w/w) to that of total proteins, a process for producing
the same, and its uses.
BACKGROUND ART
[0002] Royal jelly is a milk-white humor, secreted from cephalic
glands of worker honeybees and stored in a royal cell (a cell for
queen bee) of honeycomb, and a jelly given as a feed to larva which
grows to queen bee. Although its chemical composition is varied in
some degree depending on its source and season, it has been
reported that the composition is 65-75% (w/w) of water, 15-20%
(w/w) of proteins, 10-15% (w/w) of carbohydrates, 1.7-6% (w/w) of
fats, and 0.7-2% (w/w) of ashes. Further, royal jelly comprises
organic acids represented by 10-hydroxy-2-decenoic acid, various
vitamins and minerals. It has been reported that proteins of royal
jelly includes water-soluble proteins and water-insoluble proteins,
and the contents of the former and the later are about 75% and 25%
to the total proteins, respectively (for the example, refer to
Tetsuo Takenaka, "Mitsubachi Kagaku (Honeybee Science)", Vol. 3,
No.2, pp.69-74, 1982).
[0003] Royal jelly has long been utilized widely as a healthy food
for human. Recently, many reports have revealed that royal jelly
has useful pharmacological actions for human body such as an
antibacterial action, immune-enhancing action, anti-tumor action,
anti-inflammatory action, and life-prolonging action.
[0004] However, since royal jelly contains allergic substances, it
may cause an allergic symptom when it is consumed. Although the
allergic symptom varies depending on a person, in some cases, royal
jelly causes a significant anaphylatic shock. To overcome such
disadvantage of royal jelly, Japanese Patent Kokai No. 112,715/2002
discloses a method for reducing allergens by treating royal jelly
with saccharide-degrading enzymes and protein-degrading enzymes.
However, in the case of such royal jelly produced by the steps of
precipitating allergic water-soluble proteins by isoelectric point
precipitation and degrading the resulting precipitate by
saccharide-degrading enzymes and protein degrading enzymes, the
additional enzymes themselves would possibly be allergic
substances. Therefore, the above royal jelly has a disadvantage of
not being preferable as royal jelly for keeping health.
DISCLOSURE OF INVENTION
[0005] The present invention was accomplished under these
circumstances. The object of the present invention is to provide
ARRJ having less fear of causing allergic symptom by reducing
allergens and keeping the useful pharmacological actions of royal
jelly, a process for producing the same, and the uses of a
composition comprising ARRJ for foods, feeds, baits, pet foods, and
cosmetics.
[0006] To solve the above object, the present inventors have
extensively studied on a high quality ARRJ having less fear of
causing allergic symptom by reducing allergens and keeping the
useful pharmacological actions of royal jelly and a process for
producing AARJ. As a result, the present inventors found that the
most part of allergic substances, comprised in a fresh royal jelly,
are water-soluble and contained in supernatant by the steps of
admixing water with non-treated royal jelly (hereinafter,
"non-treated royal jelly" may be simply abbreviated as "royal
jelly" or "fresh royal jelly" in this specification. Further, it
may be abbreviated as "RJ" in Tables.) as a material, separating
the mixture into a supernatant and a precipitate by centrifugation,
and measuring the allergic activity of those using a fresh royal
jelly as a control. The present inventors also found as original
knowledge that ARRJ, where the amount of water-soluble proteins is
reduced to less than 50% (w/w) (hereinafter, "% (w/w)" is simply
abbreviated as "%" in this specification) to the amount of total
proteins and the amount of allergens is reduced, which keeps the
useful pharmacological actions of fresh royal jelly, can be
produced by a method constructed by combining the processes of
admixing water with a fresh royal jelly and separating the mixture
into a supernatant and a precipitate by centrifugation
(hereinafter, the process is abbreviated as "process of
water-fractionation" in this specification); collecting low
molecular substances by eliminating high molecular substances
comprising water-soluble proteins from the resulting supernatant
using ultra-filtration or gel-filtration chromatography
(hereinafter, the process is abbreviated as "process of high
molecular substances elimination" in this specification); and
mixing the resulting low molecular substances with the aforesaid
precipitate (hereinafter, the process is abbreviated as "process of
mixing" in this specification).
[0007] Specifically, the present invention solves the above object
by providing ARRJ, where the amount of water-soluble proteins is
reduced to less than 50% to the amount of total proteins, the
process for producing the same, a composition comprising ARRJ, and
the uses of the composition for foods, feeds, baits, pet foods, and
cosmetics.
BEST MODE FOR CARRYING OUT THE INVENTION
[0008] The present invention relates to ARRJ where the amount of
water-soluble proteins is reduced to less than 50% to the amount of
total proteins. A fresh royal jelly usable as a material for ARRJ
of the present invention is not restricted to species of bee
secreting it, sources, and its forms (raw or frozen form) supplied.
A royal jelly usable in the present invention is those secreted by
Apis mellifera, Apis cerana, Apis dorsata, Apis florae, etc. A
royal jelly usable in the present invention is obtained from a
source in Japan, South America, North America, Australia, China,
Europe, etc. Although all those royal jellies can be used as
materials for ARRJ of the present invention, it is preferable to
use royal jelly as fresh as possible or that preserved under a
relatively low temperature. In ARRJ of the present invention, since
the amount of water-soluble proteins is reduced to less than 50%,
preferably, less than 40%, more preferably, less than 20%, most
preferably, less than 10% to the amount of total proteins, the
allergic activity is reduced. In the present invention, the amount
of proteins is determined by the method of Lowry (0. H. Lowry et
al., Journal Biological Chemistry, Vol. 193, 265, 1951), a method
for protein determination used generally in the art, using bovine
serum albumin as a standard protein. While, the allergic activity
of royal jelly can be determined by the method described in the
following Experiments.
[0009] ARRJ of the present invention well keeps
10-hydroxy-2-decenoic acid having the useful pharmacological
actions of royal jelly, while high molecular substances, i.e.,
proteins, which are a cause of allergic activity, are reduced. The
weight ratio of total proteins to 10-hydroxy-2-decenoic acid, on a
free fatty acid basis, can be used for the physicochemical index of
ARRJ; and that for ARRJ of the present invention is less than six.
Since the ratio of a fresh royal jelly is usually in the range of 7
to 15, the value can be clearly discriminated from that of ARRJ of
the present invention. The amount of 10-hydroxy-2-decenoic acid can
be determined by the method described in the following
Experiments.
[0010] Also, ARRJ of the present invention can be identified by the
steps of applying the protein of ARRJ to isoelectrofocusing in the
range of pH 3-10 in the presence of 8M urea and measuring the
amount ratio of proteins showing the isoelectric point of less than
5 and that of 5 or higher. Since the value of ARRJ of the present
invention is one or higher, ARRJ can be discriminated from a fresh
royal jelly showing the value of less than one.
[0011] Various methods for purifying royal jelly can be arbitrarily
used as far as they can reduce allergic activity without
substantially losing the useful pharmacological actions of royal
jelly. Concretely, the method, where the amount of water-soluble
proteins can be reduced to less than 50%, preferably, less than
40%, more preferably, less than 20%, most preferably, less than 10%
to the amount of total proteins, is desirable. As concrete
purification methods, one or more methods generally used in the art
for purifying substances comprising proteins such as dilution with
water, centrifugation, filtration with membrane, filtration,
concentration, separatory precipitation, salting out, dialysis,
ion-exchange chromatography, gel filtration chromatography,
adsorption chromatography, chromatofocusing, hydrophobic
chromatography, reverse-phase chromatography, affinity
chromatography, gel electrophoresis, and isoelectrofocusing can be
arbitrarily used.
[0012] The process for producing ARRJ of the present invention is
constructed by the combination of processes of water-fractionation,
high molecular substance-elimination, and mixing. In the above
purification methods, dilution with water, and centrifugation are
used in a process of water-fractionation. Although membrane
fractionation and gel filtration chromatography can be used in a
process of high molecular substance-elimination, membrane
fractionation is more preferable because of its convenience.
[0013] In the present invention, water-soluble proteins are
comprised in a supernatant obtained by the process of
water-fractionation comprises the steps of admixing water to fresh
royal jelly and centrifuging. Therefore, they can be separated from
a water-insoluble precipitate. The amount of total proteins as
referred to as in the present invention means sum of the amount of
proteins in the supernatant (the amount of water-soluble proteins)
and that in the precipitate as calculated by formula 1. The content
(%) of water-soluble proteins in total proteins as referred to as
in the present invention means the value calculated by formula 2.
Total proteins =Proteins in the supernatant (Water-soluble
proteins) +Proteins in the precipitate Formula 1 The content (%) of
water-soluble proteins in total proteins =(Water-soluble
proteins/Total proteins).times.100 Formula 2
[0014] The following explains each process for purifying royal
jelly of the present invention.
Process of Water-Fractionation
[0015] As aforementioned, the present inventors found that the
allergic activity in royal jelly mainly originated from high
molecular substances which present in a supernatant obtained by
water-fractionation of royal jelly. The process is to separate
royal jelly into a supernatant and a precipitate by
water-fractionation. Water to be admixed with royal jelly is not
restricted, and optionally, any water such as ultra-pure water,
deionized water, distilled water, magnetized water, mineral water,
running water, and deep-sea water can be used. Optionally, buffers
can be used to keep pH within a prescribed range of aqueous
solution in which royal jelly is suspended or dissolved. Since the
pH of aqueous solution in which royal jelly is suspended or
dissolved is usually in the range of 3.5-4.5, it is not necessary
to control pH of the solution. However, in the case of being the pH
lower than 3.5, the solution shows relatively strong sourness and
is inconvenient for processing as foods. Also, in the case of being
the pH higher than 4.5, the solution is not preferable for
water-fractionation because inherently water-soluble proteins may
form precipitate by isoelectric point precipitation. It is
desirable to keep the pH of aqueous solution in which royal jelly
is suspended or dissolved in the range of 3.5-4.5. To stabilize
effective components comprised in royal jelly, the use of sterile
water with a relatively low temperature is desirable. Although the
amount of water to be admixed with royal jelly is varied depending
on repeating times of water-fractionation, usually, one to
1000-folds, preferably, one to 100-folds of water to the weight of
a fresh royal jelly used. In the case of using only a little amount
of water, the dissolving of water-soluble proteins will be
insufficient and the separation of a fresh royal jelly into a
supernatant and a precipitate will also be insufficient. In the
case of using too large amount of water, it causes the
deterioration of effective components and is not preferable from
the view point of working efficiency because it requires a longer
production time. Water-fractionation can be done by using a
relatively large amount of water and centrifuging at a time or by
using a relatively small amount of water and centrifuging for
several times. For the purpose of reducing the content of
water-soluble proteins in total proteins to less than 50%, it is
desirable to apply water-fractionation at least once, preferably,
twice or more. Methods for centrifugation are not restricted as far
as they can separate the suspension into a supernatant and a
precipitate well, and both batch- and continuous-modes can be
advantageously used. Although the condition of centrifuging is
varied depending on centrifugal force and running time, it is
desirable to centrifuge, usually, 3,000.times.g for 10 minutes or
longer, preferably, 5,000.times.g for 10 minutes or longer, more
preferably, 10,000.times.g for 10 minutes or longer.
Process of Eliminating High Molecular Substances
[0016] The above supernatant, showing an allergic activity,
obtained by the water-fractionation comprises water-soluble
proteins as high molecular substances. The supernatant also
comprises low molecular substances such as organic acids including
10-hydroxy-2-decenoic acid, amino acids, saccharides, and vitamins.
The process is carried out for separating substances present in the
supernatant into high molecular substances and low molecular
substances, eliminating high molecular substances comprising
water-soluble proteins, and collecting useful low molecular
substances. Water-soluble proteins as high molecular substance can
be effectively eliminated by membrane-separation using an
ultrafiltration membrane which has a pore size of not allowing to
pass water-soluble proteins. For such an ultrafiltration membrane,
having a molecular weight cut off of 1,000-10,000 daltons, is
preferable. In the case of using a membrane having a molecular
weight cut off of less than 1,000 daltons, there is a fear of being
also eliminated low molecular substances. In the case of using a
membrane having a molecular weight cut off of higher than 10,000
daltons, there is a fear of infecting a part of water-soluble
proteins as high molecular substances. Since it is possible to
separate into low molecular substances and high molecular
substances by gel filtration chromatography, such a method can also
be applied to the process of eliminating high molecular substances
of the present invention. However, with regard to working
efficiency and industrial applicability, it is desirable to use
ultrafiltration membranes.
Process of Mixing
[0017] The process is carried out for producing ARRJ of the present
invention by mixing useful low molecular substances, collected by
the process of eliminating high molecular substances, with the
aforesaid precipitate. Low molecular substances, obtained by
ultrafiltration or gel filtration, can be directly mixed with the
aforesaid precipitate. In many cases, low molecular substances,
obtained by ultrafiltration or gel filtration, are considerably
diluted in comparison with those comprised in the material. In such
case, the low molecular substances can be optionally concentrated
and mixed with the precipitate. Optionally, after mixing the low
molecular substances with the aforesaid precipitate, the mixture
can be concentrated. Methods for such concentration can be
arbitrarily selected from heating, concentrating under a reduced
pressure, and a technique using a reverse osmosis membrane. Among
these methods, concentration under a reduced pressure and that
using a reverse osmosis membrane are preferable because they have
less fear of heat-denaturing of effective components comprised in
royal jelly. In the case of concentrating with a reverse osmosis
membrane, the use of the membrane which is able to collect
water-soluble low molecular substances comprised in royal jelly to
the concentrate fraction, is preferable. It is desirable to used a
reverse osmosis membrane having a sodium chloride cut off rate of,
usually, 75% or higher, preferably, 90% or higher, more preferably,
95% or higher. AARJ of the present invention, having desirable
concentration, can be obtained by adjusting the concentration using
the procedure.
[0018] In AARJ of the present invention obtained by the above
procedure, the content of water-soluble proteins in total proteins
is reduced to less than 50%. Accompanying with the reduction, the
amount of allergic substances is reduced but the useful
pharmacological actions such as an anti-inflammatory action and
life-prolonging action, those are inherently exhibited in royal
jelly, are not lost. Therefore, AARJ of the present invention can
be advantageously used as a healthy food for the purpose of keeping
and promoting health.
[0019] While AARJ of the present invention can be used intact as
described above, it can be advantageously used in a form of
composition produced by incorporating it into other ingredients.
The present invention also provides such compositions. A
composition of the present invention usually comprises one or more
ingredients, which are allowed to use for oral- or
transdermal-applications or external use for skin, to mammals
including human. Therefore, such composition can be advantageously
used in the fields of foods, beverages, feeds, baits, pet foods,
cosmetics and the like. An ingredient useable for oral- or
transdermal-applications or external use for skin, to mammals
including human, includes substances generally used in each field
of the composition of the present invention such as water,
alcohols, starchy substances, amino acids, fibers, saccharides,
fats, fatty acids, vitamins, minerals, flavors, colorings,
sweeteners, seasonings, spices, preservatives, emulsifiers,
detergents, and the like. The form of the composition comprising
above ingredients is not restricted. Therefore, the composition of
the present invention can be provided in a desirable form such as a
powder, granule, tablet, paste, emulsion and liquid.
[0020] A composition comprising ARRJ of the present invention can
be produced by the steps of mixing above ingredients with ARRJ
based on respective content according to the object, i.e., based on
an arbitrary composition selected according to the objective mammal
and the method of administration; applying an arbitrary treatment
such as dilution, concentration, drying, filtration,
centrifugation, and the like; and optionally shaping into a
desirable form. The order of incorporating each ingredient and the
timing for applying various treatments are not restricted as far as
they do not causing the deterioration of ARRJ. For example, it is
preferable that ARRJ just after prepared or preserved under a low
temperature after preparation is incorporated into the ingredients
and then optionally applied to various treatments. For preventing
the deterioration of ARRJ in the production process, it is
preferable that the above all processes are carried out under
ambient temperature, preferably, under the condition of 30.degree.
C. or lower.
[0021] A composition comprising ARRJ of the present invention can
be processed into a solid form by incorporating it into an
anhydrous saccharide and drying. Further, ARRJ can be processed
into a form of powder, granule, tablet, and the like. As anhydrous
saccharide, anhydrous .alpha.,.alpha.-trehalose, anhydrous maltose,
anhydrous cyclic tetrasaccharide, and the like can be used.
Anhydrous .alpha.,.alpha.-trehalose can be produced easily from
"TREHA.RTM.", commercially available crystalline trehalose
dihydrate commercialized by Hayashibara Shoji Inc., Okayama, Japan,
by the method disclosed in Japanese Patent No. 3,168,550, and used
for the above object. A commercially available anhydrous
crystalline maltose, "FINETOSE.RTM.", commercialized by Hayashibara
Shoji Inc., Okayama, Japan, can be used as anhydrous maltose.
Further, anhydrous cyclic tetrasaccharide disclosed in WO
02/057,011 can be advantageously used.
[0022] A composition, produced by incorporating ARRJ into an
anhydrous saccharide of the present invention, can be obtained by
the steps of mixing ARRJ and an anhydrous saccharide, optionally,
further admixing other ingredients with the mixture, drying the
resulting mixture by allowing to absorb water to anhydrous
saccharide, and optionally further subjecting to general drying
process such as drying under reduced pressure, drying in vacuo, and
heating. More concretely, ARRJ is admixed with, usually, four-folds
or more amounts by weight, preferably, eight-folds or more amounts
by weight to the amount of ARRJ of crystalline or amorphous
anhydrous saccharide, and optionally the mixture is further admixed
with other ingredients. Successively, the resulting mixture is
dehydrated and dried by keeping under ambient temperature or lower,
preferably, 30.degree. C. or lower for, usually, four hours or
longer, preferably, eight hours or longer. Optionally, ARRJ in a
solid form, thus prepared, is further dried. ARRJ in a solid form
can be advantageously processed to a desirable form such as a
powder, granule, and tablet by using a pulverizer, granulator, and
tableting machine. Optionally, the resulting powder or granule can
be advantageously used after filling into a capsule.
[0023] Optionally, a commercially available saccharide-transferred
vitamin C (alias L-ascorbic acid 2-glucoside) can be admixed with a
composition of the present invention described above. It has been
well known that L-ascorbic acid has the function of enhancing the
production of collagen in living bodies. However, L-ascorbic acid
has disadvantages of being unstable and being easily decomposed by
oxidation. While, the saccharide-transferred vitamin C is a
chemically stable substance and hydrolyzed in living bodies to
release L-ascorbic acid.
[0024] Effective components comprised in ARRJ can be further
stabilized by incorporating antioxidant substances into a
composition of the present invention. Therefore, antioxidant
substances can be advantageously used according to the object or
area of the application, for example, in the case of transporting
the composition by ship and the like without controlling the
temperature or using the composition in an area with high
temperature. Antioxidant substances usable in the present invention
are not restricted specifically. However, in the case of using the
composition as edible one for mammal including human, it is
preferable of selecting edible antioxidant substances in the field
of foods. Concrete edible antioxidant substance, generally used in
the field of foods, includes flavonoids, polyphenols, vitamin E and
vitamin C. Flavonoids include rutin, hesperidin, naringin,
quercetin, and those derivatives, produced by transferring glucose
or its polymer (oligosaccharide), such as saccharide-transferred
rutin, saccharide-transferred hesperidin, saccharide-transferred
naringin, and saccharide-transferred quercetin. Catechin and gallic
acid can be used as polyphenols. Further, extracts of plants such
as Sophora japonic L. extract, rosemary extract, and eucalyptus
extract can be used in the present invention as antioxidant
substances. The content of these antioxidant substances in the
composition is not restricted. However, in the case of using the
composition as a food, it is preferable to use it according to the
amount generally used in the field of foods or less than that.
[0025] A desirable form of the composition of the present invention
as foods includes, for example, frozen dessert such as ice cream,
ice candy, and sherbet; syrup such as "korimitsu" (a sugar syrup
for shaved ice), spreads and pastes such as butter cream, custard
cream, flour paste, peanut paste, and fruit paste; confectionery
such as chocolate, jelly, candy, gummy jelly, caramel, chewing gum,
custard pudding, cream puff, sponge cake, and the like; processed
fruit or vegetable such as jam, marmalade, "syrup-zuke" (fruit
pickles), and "toka" (conserves), and the like; Japanese
confectionery such as "manju" (a bun stuffed with azuki-bean
paste), "uiro" (a sweet rice jelly), "an" (azuki-bean jam), "yokan"
(a sweet bean jelly), "mizuyohkan" (a soft sweet bean jelly), pao
de Castella, "amedama" (a Japanese toffee), and the like; and
seasoning such as soy-sauce, powderized soy-sauce, "miso" (soy-bean
paste), powdered "miso", mayonnaise, dressing, vinegar, "sanbai-zu"
(a sauce of sugar, soy sauce and vinegar), table sugar, coffee
sugar, and the like. Desirable form as beverage of the composition
of the present invention includes, for example, alcoholic beverage
such as synthetic sake, fermented liquor, fruit liquor, and sake;
and soft drink such as juice, beverage containing minerals,
carbonated beverage, sour milk beverage, beverage containing a
lactic acid bacterium, isotonic drink, nutritional drink,
green-tea, tea, oolong tea, coffee, cocoa, and the like.
[0026] ARRJ of the present invention can be also advantageously
used intact or in the form of composition comprising other
ingredients for a feed, bait, pet food for animals such as domestic
animals, poultry, pets, honeybees, silk warms, insects, and fishes.
As other ingredients which can be incorporated into compositions
having such forms, one or more ingredients, usually used in the
respective fields of feeds, baits, and pet foods, for example,
grains, starchy substances, starch hydrol, saccharides, fats, nuts,
beans, fish and shellfish, meats, eggs, milks, extract of plant
protein, fruits, mushrooms, algae, vitamins, minerals, amino acids,
yeast, grasses, bagasse, corncob, rice straw, hays, oil cakes,
bran, soybean-bran, and various fermented bran can be used. The
composition can be advantageously used in solid form such as
powder, granule, tablet, paste, gummy, and the like, and liquid
form such as emulsion and drink.
[0027] A desirable form of the composition of the present invention
as cosmetics includes basic skin care cosmetics, cosmetics for
washing, cosmetics for bath, hair care cosmetics, suntan and
sunscreen cosmetics, makeup cosmetics, hair growth stimulants, and
hair growth tonics.
[0028] To produce a composition of the present invention in the
form of above, ARRJ of the present invention can be incorporated
into the composition at arbitrary timing in the process of
producing the objective product according to the conventional
method. The timing of incorporating ARRJ is not specifically
restricted. However, in the case of producing the objective product
through a heating process, the decreasing of the useful
pharmacological action of ARRJ in the production process can be
prevented by incorporating ARRJ into the product after cooling that
to ambient temperature, preferably, 30.degree. C. or lower after
the heating process. A composition of the present invention
described above comprises ARRJ of the present invention, usually,
0.001-20%, preferably, 0.01-10%, on a weight of product basis.
[0029] In the composition of the present invention described above,
since the allergic activity is reduced and various useful
pharmacological actions are stabilized, the useful pharmacological
actions are effectively expressed in living bodies and the
composition can be used for increasing resistance of living bodies,
improving bad condition quickly, and keeping good health.
Accordingly, the composition of the present invention is very
useful as foods, beverages, feeds, baits, pet foods, cosmetics, and
the like.
[0030] The following experiments explain the present invention in
detail:
Experiment 1
Water-Fractionation of Brazilian and Chinese Royal Jellies and Test
for Allergic Activity
Experiment 1-1
Water-Fractionation of Brazilian and Chinese Royal Jellies
[0031] Brazilian fresh royal jelly (moisture content of 62.5%) and
Chinese fresh royal jelly (moisture content of 65.7%), those are
frozen and preserved, were thawed and weighed 12 grams each.
Forty-eight grams, four-folds amount by weight to the weighed royal
jelly, of sterile deionized water was admixed with above royal
jelly and stirred to homogeneity. After confirming the uniform
dispersion, the suspension was centrifuged at 5,000.times.g for 10
minutes, and the resulting supernatant and precipitate were
collected, respectively. As a control, 12 grams of a fresh royal
jelly and 48 grams of water were mixed, and then stirred and
dispersed similarly as above.
Experiment 1-2
Test for Allergic Activity
[0032] On the evaluation of allergic activity comprised in a
supernatant and a precipitate prepared from Brazilian and Chinese
royal jellies in Experiment 1-1, the amount of all samples was used
by adjusting the amount corresponding to fresh royal jelly. The
amount corresponding to fresh royal jelly as referred to as in the
present invention means the amount of sample converted to the
weight of material fresh royal jelly, and the amount of sample
originated from one gram of inherent fresh royal jelly is expressed
to one gram corresponding to fresh royal jelly in each case of
supernatant or precipitate. Test for allergic activity was carried
out using an antigenicity test for mice according to the method of
Hagita et al. (Tadaatsu Hagita and Hiroshi Mizushima, "Igaku no
Ayumi (Proceeding of Medical Science)", Vol. 100, pp. 814, 1977).
Specifically, 1.5 mg corresponding to fresh royal jelly of each
sample and 2.5 mg of aluminum hydroxide gel (Alum) were
administrated to peritoneal cavity of five female BALB/c mice
(nine-weeks-old, commercialized by Charles River Japan Inc.,
Kanagawa, Japan) per group with three times every other week. One
week later from the third administration, blood was collected from
the caudal vein of each mouse and then serum was collected using a
centrifuging tube and subjected to PCA method. Specifically, 0.1 ml
each of mouse serum diluted to 100- or 200-folds with saline was
used for intradermal injection to the dehaired-back of SD mouse.
After 24 hours from the injection, 7.5 mg corresponding to fresh
royal jelly of preparation identical with that administrated to
mouse and one ml of 0.5% Evan's Blue solution were used for
intravenous injection, and the size of blue spot observed at the
position of injecting serum was measured. A mouse, which shows the
spot having an average diameter of 5 mm or higher, was judged as
"positive". The strength of allergic activity was estimated to be
"+++++" where all five mice in a test group are "positive" and "-"
where all five mice are "negative". The results for supernatant,
precipitate, and control of Brazilian royal jelly and those of
Chinese royal jelly were in Table 1 and 2, respectively.
TABLE-US-00001 TABLE 1 Allergic activity Serum-dilution factor
100-folds 200-folds Control (Fresh royal jelly) +++ - Supernatant
+++++ ++ Precipitate + -
[0033] TABLE-US-00002 TABLE 2 Allergic activity Serum-dilution
factor 100-folds 200-folds Control (Fresh royal jelly) +++++ +++
Supernatant +++++ +++++ Precipitate ++++ +
[0034] As is evident from the results in Table 1 and 2, a
relatively strong allergic activity was detected in a supernatant
in both cases of Brazilian royal jelly and Chinese royal jelly. On
the contrary, allergic activities in precipitate were relatively
weak. It was supposed that water-soluble proteins were major
allergic substances. Further, from the comparison of the results
obtained by serum-dilution factor of 100- and 200-folds, it was
revealed that the allergic activity of Chinese royal jelly is
stronger than that of Brazilian royal jelly.
Experiment 2
Effects of Repeating Time of Water-Fractionation on the Content of
Water-Soluble Proteins to the Total Proteins Recovered in
Precipitate by Water-Fractionation and the Reduction of Allergic
Activity
Experiment 2-1
Effects of Repeating Time of Water-Fractionation on the Content of
Water-Soluble Proteins to the Total Proteins Recovered in
Precipitate by Water-Fractionation
[0035] To examine the effects of repeating time of
water-fractionation on the content of water-soluble proteins to the
total proteins recovered in precipitate, 12 grams of Brazilian
royal jelly (moisture content of 62.5%) was used as material.
Similarly to the case of Experiment 1-1, four-folds amount by
weight (48 grams) of water was admixed with the material and the
resulting mixture was separated into a supernatant and a
precipitate by centrifuging at 5,000.times.g for 10 minutes. To the
resulting precipitate, same water-fractionation procedure was
repeated two-times to prepare precipitate. In order to calculate
the content of water-soluble proteins comprised in each precipitate
obtained by one-, two-; or three-times water-fractionation,
four-folds amount by weight of water was admixed with each
precipitate similarly to the above water-fractionation. After
centrifuging, the amount of proteins in the supernatant and that in
precipitate were determined by the method of Lowry et al. using
bovine serum albumin as a standard protein. The amount of proteins
in the supernatant was defined as the amount of water-soluble
proteins. As a control, a fresh royal jelly suspension prepared as
in the case of Experiment 1-1 was centrifuged and the amount of
proteins was determined in a same manner with above precipitate.
Assay data of total proteins and water-soluble proteins comprised
in precipitate recovered from each water-fractionation times and
the content of water-soluble proteins in total proteins calculated
from those data are in Table 3. TABLE-US-00003 TABLE 3 Water-
Proteins in Proteins in Total Content of fractionation supernatant
precipitate proteins WSP* (Times) (mg) (mg) (mg) (%) Control 1092.0
567.0 1569.0 65.8 (Fresh RJ**) 1 272.0 424.8 696.8 39.0 2 25.9
412.8 438.7 5.9 3 16.5 387.6 404.1 4.1 *Water-soluble proteins
**Royal jelly
[0036] As is evident from the results in Table 3, the content of
water-soluble proteins to total proteins in fresh royal jelly was
65.8%. While, the values of the precipitates obtained by one- and
two-times water-fractionation were reduced to about 40% and about
6%, respectively.
Experiment 2-2
The Relationship Between the Precipitate Obtained by
Water-Fractionation and its Allergic Activity
[0037] Allergic activity of precipitates, obtained by the
water-fractionation of one-, two-, and three times in Experiment
2-1, was determined by the same method of Experiment 1-2 except for
altering the serum-dilution factor to 50- and 100-folds. A
suspension of fresh royal jelly prepared by the same method of
Experiment 1-1 was used as control. The results are in Table 4.
TABLE-US-00004 TABLE 4 Allergic activity Water-fractionation
Serum-dilution factor (Times) 50-folds 100-folds Control (RJ*)
+++++ +++ 1 ++ + 2 + - 3 - - *Royal jelly
[0038] From above results, it was revealed that the content of
water-soluble proteins in total proteins of precipitate can be
reduced to less than about 40% and the allergic activity can be
remarkably reduced by applying the water-fractionation once.
Experiment 3
Preparation of Various Preparations from Royal Jelly, and ARRJ
[0039] Sixty grams of Chinese fresh royal jelly as material and 240
grams of deionized water at 4.degree. C. were mixed and stirred to
give homogenous dispersion. Successively, the suspension was
centrifuged (5,000.times.g for 10 minutes) and separated into a
supernatant and a precipitate. The resulting precipitate was mixed
with 240 grams of deionized water at 4.degree. C. again and treated
the suspension as above to obtain a precipitate. A supernatant of
water-fractionation was prepared by mixing a supernatant obtained
by the first water-fractionation and that by the second and
subjected to ultrafiltration (UF) using "AIP0013", a pencil type
ultrafiltration membrane having a molecular weight cut off of 6,000
daltons, commercialized by Asahi Kasei Corporation, Tokyo, Japan.
The supernatant was separated into a UF-filtrate and a
UF-concentrate. Finally, ARRJ was prepared by mixing the resulting
UF-filtrate and the aforesaid precipitate. The amounts of
10-hydroxy-2-decenoic acid, proteins and saccharides comprised in
samples of each preparation step were determined.
10-Hydroxy-2-decenoic acid is a specific component of royal jelly
as an index of effective component and reported to be involving
saccharide metabolism and prevention of aging. The amount of
proteins was determined by the method of Lowry et al. using bovine
serum albumin as a standard protein. The amount of saccharides was
determined by anthrone-sulfuric acid method using glucose as a
standard saccharide. The amount of 10-hydroxy-2-decenoic acid was
determined by HPLC method using the following conditions.
HPLC Conditions for the Determination of 10-Hydroxy-2-Decenoic
Acid
[0040] Column: YMC-Pack AQ-303-ODS (.phi. 4.6 mm.times.250 mm, YMC
Co., Ltd.) [0041] Column temperature : 40.degree. C. [0042]
Detection : UV 210 nm [0043] Mobile phase : 50% methanol (pH was
adjusted to 2.2 by phosphoric acid) [0044] Flow rate: 0.6 ml/minute
[0045] Sample volume : 20 .mu.l The amount of 10-hydroxy-2-decenoic
acid was determined by the standard curve prepared using standard
10-hydroxy-2-decenoic acid solutions of 20, 50, and 100
.mu.g/ml.
[0046] Recovery (%) of each component at each preparation step was
calculated using their amounts comprised in material fresh royal
jelly as 100%, respectively. The results are in Table 5.
TABLE-US-00005 TABLE 5 Weight of Preparation solution Proteins
10-HDA**** Saccharides No. Preparation (g) (mg) Yield (%) (mg)
Yield (%) (mg) Yield (%) 1 Fresh RJ* 60 8280 100 1145 100 8580 100
2 Precipitate 275 2305 28 275 24 165 2 3 Supernatant 500 6605 80
800 70 8620 100 4 UF**-filtrate 600 195 2 660 58 7820 91 5
UF-concentrate 250 5830 70 5 1 915 11 6 ARRJ*** 875 2500 30 935 82
7985 93 *Royal jelly **Ultra-filtration ***Allergen-reduced royal
jelly of the present invention ****10-hydroxy-2-decenoic acid
[0047] As is evident from Table 5, water-soluble proteins, which
account for major part, 6,605 grams, of proteins comprised in royal
jelly, were moved to a supernatant by water-fractionation, and it
was revealed that 5,830 grams of water-soluble proteins, about 90%
of total water-soluble proteins, can be eliminated as a
UF-concentrate. It is also revealed that saccharides and useful low
molecular substances represented by 10-hydroxy-2-decenoic acid,
comprised in material fresh royal jelly were moved to supernatant
together with water-soluble proteins having a relatively strong
allergic activity, and can be recovered in ARRJ of the present
invention which is produced by mixing the precipitate and the
UF-filtrate with the recovery of about 90% or higher and 80% or
higher by the steps of applying the supernatant to UF-filtration,
collecting the resulting filtrate, and admixing the filtrate with
the precipitate. The weight ratio of total proteins to that of
10-hydroxy-2-decenoic acid, on a free-fatty acid basis, was about
2.7. The value was clearly different from that of material fresh
royal jelly of about 7.2.
Experiment 4
Allergic Activity and Useful Pharmacological Action of Various
Preparations from Royal Jelly and ARRJ
[0048] Allergic activity and activity for suppressing the
production of tumor necrosis factor-.alpha. (TNF-.alpha.), an index
of anti-inflammatory action, of various preparations obtained from
royal jelly in Experiment 3 and ARRJ were investigated.
Experiment 4-1
Determination of Activity for Suppressing the Production of Tumor
Necrosis Factor-.alpha. (TNF-.alpha.)
[0049] 1.5 milliliters of 4% thioglycolate medium was injected into
the abdominal cavity of BALB/c mouse. After three days, peritoneal
macrophages induced in BALB/c mouse were collected from the
abdominal cavity. Successively, each preparation obtained in
Experiment 3-1 was added to a medium with adjusting the amount to
that corresponding to material fresh royal jelly, and
lipopolysaccharide (LPS), interferon-.gamma. (IFN-.gamma.), and
BALB/c mouse peritoneal macrophage were admixed with the medium to
give final concentrations of 1 .mu.g/ml, 10 IU/ml, and
5.times.10.sup.5 cells/well, respectively, and the mixture was put
in a well. After cultivating for 48 hours, the resulting culture
supernatant was collected and the amount of TNF-.alpha. produced
was measured by solid-phase enzyme-linked immuno solvent assay
(ELISA). As a control 1, the measurement was carried out by the
same procedure using phosphate-buffer saline instead of various
preparations from royal jelly. As a control 2, the measurement was
carried out with the same manner using a fresh royal jelly with the
same concentration. The relative activity for suppressing the
production of TNF-.alpha. was represented by percentage using the
difference of TNF-.alpha. activities of control 1 and 2 as 100.
Allergic activities of each preparation from royal jelly and ARRJ
were evaluated according to the method in Experiment 1-2. The
results are in Table 6. TABLE-US-00006 TABLE 6 Allergic activity
Relative activity (serum dilution for suppressing Preparation
factor: 200- the production of No. Preparation folds) TNF-.alpha.
(%) 1 Fresh RJ* +++ 100 2 Precipitate + 0 3 Supernatant +++++ 100 4
UF**-filtrate - 90 5 UF-concentrate +++++ 10 6 ARRJ*** + 90 *Royal
jelly **Ultrafiltration ***Allergen-reduced royal jelly of the
present invention
[0050] As is evident from the results in Table 6, substances
showing an allergic activity were moved to UF-concentrate by
water-fractionation and membrane separation. The allergic activity
of ARRJ which is prepared by mixing the precipitate of
water-fractionation and the UF-filtrate was reduced to 1/3 compared
with that of material fresh royal jelly. Further, it was revealed
that about 90% of activity for suppressing the production of
TNF-.alpha., which is an index of anti-inflammatory inherently
exhibited in royal jelly, was recovered in ARRJ of the present
invention from material fresh royal jelly. As described above, it
was concluded that AARJ of the present invention is useful for
keeping and increasing the health because the allergic activity
inherently exhibited in royal jelly was reduced and whose useful
pharmacological actions were kept well.
Experiment 5
Comparison of Proteins Comprised in ARRJ and Fresh Royal Jelly by
Gel Isoelectrofocusing
[0051] To compare proteins comprised in ARRJ prepared by the method
of Experiment 3 with those comprised in material Chinese royal
jelly, both samples were subjected to gel isoelectrofocusing. Forty
micrograms each of ARRJ and fresh royal jelly was put into a test
tube and dried in vacuo. The resultant was dissolved in 100 .mu.l
of a solution for isoelectrofocusing comprising 8 M urea, 4% (w/v)
CHAPS, and 0.5% (w/v) "Pharmalyte.RTM.", a carrier ampholyte
commercialized by Amersham Biosciences K. K., Tokyo, Japan, and
then admixed with 150 .mu.l of a solution comprising 8 M urea, 4%
(w/v) CHAPS, 50 mM dithiothreitol, and 1% IPG buffer commercialized
by Amersham Biosciences K. K., Tokyo, Japan, to prepare a sample
for electrophoresis. Successively, the sample was put in an
exclusive container and "IMMOBILINE DryStrip, pH 3-10, 13 cm", a
precast gel for isoelectrofocusing commercialized by Amersham
Biosciences K. K., Tokyo, Japan, was soaked in the sample solution.
The gel was swelled by allowing to absorb the sample with keeping
at room temperature for 16 hours. Isoelectrofocusing was carried
out using the resulting gel with a condition of 100 V for two
hours, 300 V for five hours, and 3,500 V for 12 hours. After the
isoelectrofocusing, proteins separated in the gel were stained by
soaking the gel in a solution comprising 0.025% (w/v) Coomassie
Brilliant Blue (CBB) R-250, 40% (v/v) methanol, and 7% (v/v) acetic
acid. Successively, the stained gel was destained by stirring in a
solution comprising 40% (v/v) methanol and 7% (v/v) acetic acid for
30 minutes and that comprising 5% (v/v) methanol and 7% (v/v)
acetic acid for five hours at room temperature.
[0052] Proteins, comprised in ARRJ or fresh royal jelly were
roughly separated into two protein groups with a borderline of
isoelectric point 5. Therefore, the percentages (%) of proteins
showing an isoelectric point of lower than 5 and that of 5 or
higher were compared by the steps of analyzing the destained gel
with "DUAL-WAVELENGTH CHROMATO SCANNER CS-930", a gel scanner
commercialized by Shimadzu Corporation, Kyoto, Japan, and measuring
the area ratio of absorption peak at 600 nm. The average results of
four independent tests for ARRJ and fresh royal jelly are in Table
7, respectively. TABLE-US-00007 TABLE 7 Percentage of protein (%)*
Sample pI** lower than 5 pI 5 or higher ARRJ (present invention)
43.3 56.7 Fresh royal jelly 58.3 41.7 *Average of four independent
tests **Isoelectoric point
[0053] As is evident from the results shown in Table 7, in the
usual fresh royal jelly, the percentage of proteins showing
isoelectric point of 5 or higher was about 42% of total proteins.
While, that of ARRJ of the present invention was about 57%. The
results suggest that water-soluble proteins, having a strong
allergic activity, which are reduced by water-fractionation,
comprise relatively large amount of proteins having isoelectric
point of lower than 5. The feature of ARRJ of the present invention
is the fact that the ratio of proteins showing isoelectric point of
5 or higher to that of lower than 5 is one or higher at gel
isoelectrofocusing, which is carried out in the presence of 8 M
urea and in the range of pH 3-10, described in this
specification.
[0054] Although the following examples concretely explain the
present invention, the present invention is not restricted by
them:
EXAMPLE A-1
Preparation of ARRJ
[0055] Ten kilograms of a Brazilian fresh royal jelly was mixed
with 40 kilograms of deionized water with a temperature of
4.degree. C. and stirred to gave a homogenous dispersion.
Successively, the suspension was continuously centrifuged at
10,000.times.g and at flow rate of 10 L/hour using a centrifugal
separator "Model KT-2000G", commercialized by Kubota Manufacturing
Corporation, Tokyo, Japan, to obtain a supernatant and a
precipitate. The resulting supernatant was subjected to
ultrafiltration using "AIP 2013", a UF-membrane having a molecular
weight cut off of 6,000 daltons, commercialized by Asahi Kasei
Corporation, Tokyo, Japan. The supernatant was separated into a
UF-filtrate and a UF-concentrate using a small amount of deionized
water as a dilution solvent. About 40 L of the resulting
UF-filtrate was concentrated to give a weight of about 5 kilograms
using "HOLLOSEP.RTM.", a reverse osmosis membrane commercialized by
Toyobo Co., Ltd., Osaka, Japan, at a pressure of 50 kgf/cm.sup.2.
The resulting concentrate was admixed with the aforesaid
precipitate obtained by the centrifugation and stirred to
homogeneity to produce ARRJ. In ARRJ, the weight ratio of total
proteins to 10-hydroxy-2-decenoic acid, on a free-fatty acid basis,
was 3.7. By the analysis with gel isoelectrofocusing, the amount
ratio of proteins showing an isoelectric point of 5 or higher to
that of lower than 5 was about 1.3.
EXAMPLE A-2
Preparation of ARRJ
[0056] Twenty kilograms of a Chinese fresh royal jelly was mixed
with 80 kilograms of deionized water with a temperature of
4.degree. C. and stirred to gave a homogenous dispersion.
Successively, the suspension was continuously centrifuged at
10,000.times.g and at flow rate of 10 L/hour using a centrifugal
separator "Model KT-2000G", commercialized by Kubota Manufacturing
Corporation, Tokyo, Japan, to obtain a supernatant and a
precipitate. The resulting precipitate was admixed with 80
kilograms of deionized water with a temperature at 4.degree. C.
again and treated with same manner described above to obtain a
precipitate. The first and the second supernatant obtained by above
water-fractionation were mixed and the resulting solution was
subjected to ultrafiltration using "AIP 2013", a UF-membrane having
a molecular weight cut off of 6,000 daltons, commercialized by
Asahi Kasei Corporation, Tokyo, Japan. The solution was separated
into a UF-filtrate and a UF-concentrate using a small amount of
deionized water as a dilution solvent. About 160 L of the resulting
UF-filtrate was concentrated to give a weight of about 12 kilograms
using "HOLLOSEP.RTM.", a reverse osmosis membrane commercialized by
Toyobo Co., Ltd., Osaka, Japan, at a pressure of 50 kgf/cm.sup.2.
The resulting concentrate was admixed with the aforesaid
precipitate obtained by the centrifugation and stirred to
homogeneity to produce ARRJ. In ARRJ, the weight ratio of total
proteins to 10-hydroxy-2-decenoic acid, on a free-fatty acid basis,
was 4.2. By the analysis with gel isoelectrofocusing, the amount
ratio of proteins showing an isoelectric point of 5 or higher to
that of lower than 5 was about 1.2.
EXAMPLE A-3
Preparation of ARRJ
[0057] Five parts by weight of a Chinese fresh royal jelly was
mixed with 20 parts by weight of deionized water with a temperature
of 4.degree. C and stirred to gave a homogenous dispersion.
Successively, the suspension was continuously centrifuged at
10,000.times.g and at flow rate of 10 L/hour using a centrifugal
separator "Model KT-2000G", commercialized by Kubota Manufacturing
Corporation, Tokyo, Japan, to obtain a supernatant and a
precipitate. The resulting precipitate was admixed with 20 parts by
weight of deionized water with a temperature at 4.degree. C. again
and treated with same manner described above to obtain a
precipitate. The first and the second supernatant obtained by above
water-fractionation were mixed and the resulting solution was
subjected to gel filtration chromatography using "TOYOPEARL HW-40F"
gel, a gel for gel filtration commercialized by Tosoh Corporation,
Tokyo, Japan. The components were separated into a high
molecular-fraction and a low molecular-fraction by eluting with
deionized water. The resulting low molecular-fraction was
concentrated to give eight parts by weight using "Model
RE-10E-100", a reduced-pressure evaporator commercialized by
Shibata Scientific Technology, Ltd., Tokyo, Japan, at 40.degree. C.
The resulting concentrate was admixed with the aforesaid
precipitate obtained by the centrifugation and stirred to
homogeneity to produce ARRJ. In ARRJ, the weight ratio of total
proteins to 10-hydroxy-2-decenoic acid, on a free-fatty acid basis,
was 4.9. By the analysis with gel isoelectrofocusing, the amount
ratio of proteins showing an isoelectric point of 5 or higher to
that of lower than 5 was about 1.25.
REFERENTIAL EXAMPLE
Preparation of Anhydrous .alpha.,.alpha.-trehalose
[0058] "TREHA.RTM.", a hydrous .alpha.,.alpha.-trehalose product
commercialized by Hayashibara Shoji Inc., Okayama, Japan, was dried
under reduced pressure under the condition of at 90.degree. C. and
barometric pressure at -300 to -350 mmHg for about seven hours
using a rotary vacuum dryer with a jacket. After drying, anhydrous
.alpha.,.alpha.-trehalose was obtained by the steps of reducing the
temperature to ambient temperature, returning the barometric
pressure to atmospheric condition, and collecting the product.
EXAMPLE B-1
Healthy Food
[0059] Five parts by weight of anhydrous .alpha.,.alpha.-trehalose
obtained by the method of Referential example and one part by
weight of ARRJ obtained by the method of Example A-1 were mixed to
homogeneity for 15 minutes using "MODEL MDR-60", a multipurpose
mixer commercialized by Dalton Corporation, Tokyo, Japan. After
drying the mixture in vacuo at 40.degree. C. for overnight, the
resultant was pulverized using "POWERMILL P-3", a mill
commercialized by Dalton Corporation, Tokyo, Japan, with a screen
size of 0.5 mm.
[0060] The powdery composition was shaped into tablets of about 200
mg/tablet using a tableting machine. The product is a composition
which can be used easily and has a remarkable potency. Since the
product shows a mild sweetness and moderate sourness, it is a
useful healthy food for daily use.
EXAMPLE B-2
Healthy Food
[0061] A composition, comprising ARRJ of the present invention in a
powdery form, was prepared by the steps of mixing the following
ingredients with following proportion and processing the mixture
according to the procedure in Example B-1. After the preparation,
it was confirmed by the experiment according to Experiment 1 that
the composition has a reduced allergic activity. TABLE-US-00008
Anhydrous .alpha.,.alpha.-trehalose obtained by the method of 8.5
parts by weight Referential example ARRJ obtained by the method of
Example A-2 0.5 part by weight ".alpha.G-HESPERIDIN PS", a
saccharide-transferred 0.5 part by weight hesperidin commercialized
by Hayashibara Shoji Inc., Okayama, Japan "PULLULAN PF-20", a
pullulan product 0.5 part by weight commercialized by Hayashibara
Shoji Inc., Okayama, Japan
[0062] The composition comprising ARRJ was shaped into tablets of
about 300 mg/tablet using a tableting machine. The product is a
composition where the allergic activity is reduced and has a
remarkable potency. Since the product shows a mild sweetness and
moderate sourness, it is a useful healthy food for daily use.
EXAMPLE B-3
Healthy Food
[0063] A composition, comprising ARRJ of the present invention in a
powdery form, was prepared by the steps of mixing the following
ingredients with following proportion and processing the mixture
according to the procedure in Example B-1. After the preparation,
it was confirmed by the experiment according to Experiment 1 that
the composition has a reduced allergic activity. TABLE-US-00009
Anhydrous .alpha.,.alpha.-trehalose obtained by the method of 7.5
parts by weight Referential example ARRJ obtained by the method of
Example A-3 1.0 part by weight Maltitol 1.3 parts by weight
L-Tryptophan 0.2 part by weight
[0064] The product is a composition comprising ARRJ where the
allergic activity is reduced and has a remarkable potency. Since
the product shows a good taste with mild sweetness and moderate
sourness, it is a useful healthy food for daily use.
EXAMPLE B-4
Ice Cream
[0065] Eighteen parts by weight of fresh cream (fat content; about
46% (w/w)), seven parts by weight of skim milk, 51 parts by weight
of milk, 10 parts by weight of sucrose, four parts by weight of
"NYU-KA OLIGO.RTM.", a powdery product comprising lactosucrose, two
parts by weight of pullulan, and two parts by weight of gum arabic
were mixed and dissolved. After sterilizing the mixture by keeping
at 70.degree. C. for 30 minutes, the resultant was emulsified and
dispersed using a homogenizer, then rapidly cooled to 3-4.degree.
C. and admixed with four parts by weight of ARRJ obtained by the
method of Example A-2. After aging the mixture for one night, ice
cream was obtained by freezing in a freezer.
[0066] The product is an ice cream showing a moderate sweetness and
fine flavor and being useful for keeping and increasing health.
EXAMPLE B-5
"Amazake"
[0067] Ten parts by weight of polished rice was admixed with water
and boiled according to the conventional method. Successively, the
boiled rice was cooled to 55.degree. C. and admixed with 30 parts
by weight of "koji" prepared by the conventional method and 0.1
part by weight of sodium chloride and then kept at 50 to 55.degree.
C. for eight hours. After homogenizing the mixture using a mixer
and cooling to about 25.degree. C., two parts by weight of ARRJ
obtained by the method of Example A-1 was admixed with the
homogenate, stirred, and packed into a package to produce
"amazake".
[0068] The product is a high-quality "amazake" having a
satisfactory color and flavor. Further, it is a useful drink for
keeping and increasing health.
EXAMPLE B-6
Healthy Beverage
[0069] A composition was produced by mixing 500 parts by weight of
"FINETOSE.RTM.", an anhydrous crystalline maltose product, 100
parts by weight of ARRJ obtained by the method of Example A-1, 190
parts by weight of powderized egg yolk, 200 parts by weight of skim
milk, two parts by weight of "AA2G.RTM.", a saccharide-transferred
vitamin C commercialized by Hayashibara Biochemical Laboratories
Inc, Okayama, Japan, 4.4 parts by weight of sodium chloride, 1.85
parts by weight of potassium chloride, four parts by weight of
magnesium sulfate, 0.01 part by weight of thiamin, 0.1 part by
weight of L-ascorbic acid sodium salt, 0.6 parts by weight of
vitamin E acetate, and 0.04 part by weight of nicotinamide.
Twenty-five parts by weight of the composition was admixed with 150
parts by weight of purified water, dissolved to give homogenous
dispersion and enclosed 150 grams each in a brown glass bottle.
[0070] Since the product has a reduced allergic activity and is
supplemented with nutrients, it is advantageously used as a healthy
beverage for the purpose of keeping health, promoting growth,
preventing disease, promoting cure, promoting the recovery from
distress after sports. The product can be advantageously used as a
composition for oral intake or per-tube administration for animals
such as domestic animals as well as human.
EXAMPLE B-7
Pet Food (Gummy)
[0071] Thirty parts by weight of sucrose was admixed with eight
parts by weight of water and dissolved by heating. After mixing the
resultant with 50 parts by weight of starch syrup and boiled to
give a Brix value of 85 to 90.degree., the mixture was cooled to
80.degree. C. or lower. Successively, a solution, prepared by
mixing seven parts by weight of gelatin and ten parts by weight of
water and dissolving by heating, was admixed with above saccharide
solution. Successively, the mixture was further admixed with two
parts by weight of beef extract, three parts by weight of 50% (w/w)
citric acid aqueous solution, one part by weight of ARRJ obtained
by the method of Example A-1, and suitable amount of flavor, and
stirred to homogeneity. The composition thus obtained was poured
into a starch mold and solidified by preserving for one night to
produce a pet food in a gummy form.
[0072] Since the product comprises ARRJ where the allergic activity
is reduced and is supplemented with nutrients, it ca be
advantageously used as a pet food for the purpose of keeping
health, promoting growth, preventing disease, and promoting cure of
pets. The product can be advantageously used as a composition for
oral intake for animals such as various pets and domestic animals
as well as dogs and cats.
EXAMPLE B-8
Skin Cream for External Use
[0073] The following ingredients were conventionally mixed with
heating according to the following proportion. TABLE-US-00010
Polyoxyethlene-glycerin monostearate 2.0 parts by weight
Self-emulsifying glycerin monostearate 5.0 parts by weight
Eicosanyl behenoate 1.0 part by weight Liquid paraffin 1.9 parts by
weight Trimethyrolpropane trioctanoate 10.0 parts by weight
[0074] The following ingredients except for ARRJ were admixed with
the above mixture according to the following proportion and cooled
to 30.degree. C. or lower. Further, ARRJ was admixed with the
resultant according to the following proportion and emulsified
using a homogenizer to produce skin cream for external use.
TABLE-US-00011 1,3-Butyleneglycol 5.0 parts by weight Aqueous
solution of sodium lactate 10.0 parts by weight Methyl
paraoxybenzoate 0.1 part by weight Peach-leaf extract 1.5 parts by
weight Purified water 62.2 parts by weight ARRJ obtained by the
method of Example A-3 1.0 part by weight
[0075] Since the cream has a satisfactory moisture-retaining
ability and allergic activity of royal jelly was reduced, there is
no fear of causing allergic symptoms. Therefore, the cream is
useful as a fundamental cosmetic for keeping freshness of skin.
INDUSTRIAL APPLICABILITY
[0076] As is evident from the above, the present invention is based
on an original knowledge that ARRJ whose water-soluble protein
content in total proteins is reduced to less than 50% shows a
remarkably low allergic activity and keeps the useful
pharmacological action of royal jelly. Since ARRJ has no fear of
causing significant allergic symptoms, it can be used by mammals
including human easily and comfortably for keeping and increasing
their health. Also, ARRJ of the present invention, having the above
merits, can be advantageously used as various compositions such as
foods, beverages, feeds, baits, pet foods, and cosmetics by
incorporating with other ingredients.
[0077] The present invention has a remarkable effectiveness
described above and is a significantly important invention that
greatly contributes to this art.
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